Members of the TNF/NGF receptor family are expressed in almost all types of cells and control a wide range of diverse cellular activities. They have the ability both to induce cellular changes that are protein-synthesis independent, the best known of which is caspase-mediated cell death (the extrinsic cell-death pathway), and to modulate gene-expression patterns both on the transcriptional and the post-transcriptional levels. These effects contribute to the control of practically all aspects of immune defense as well as some embryonic-development and tissue-homeostatic processes. They vary, and depending on the type of cell and the identity of the activated receptor, as well as on numerous other determinants, some effects might even oppose others. This wide range of activities is mediated by a rather small number of signaling proteins, of which the best characterized are two death-domain-containing adapters, FADD/MORT1 and TRADD, the inducer caspases caspase-8 and -10, members of the TRAF ring-finger proteins, and cellular inhibitor of apoptosis protein 1 (cIAP1) and cIAP2 (ring-finger proteins with IAP motifs) (Wallach et al., Annu Rev Immunol. 1999;17:331-67. Review.) (Locksley et al., Cell. 2001 Feb. 23; 104(4):487-501.). How this limited set of proteins mediates the multiplicity of different effects of the receptors, and how the nature of the induced effect is adjusted to need, are still poorly understood.
SIVA, an additional protein suggested to participate in the proximal signaling activities of members of the TNF/NGF receptor family, was identified by virtue of its binding to the receptor CD27 in the yeast two-hybrid test (Prasad et al., 1997). Some evidence was also presented for its association with several other members of the TNF/NGF receptor family (Nocentini and Riccardi, 2005). The existence of SIVA has been known for some years, and it was shown that when overexpressed for prolonged periods this protein kills cells (Prasad et al., 1997). However, whether this is its genuine and sole activity is not known. SIVA shows no close structural resemblance to any other known protein. One region within it that initially appeared to resemble the death domain does not contain the structural signatures by which that domain is characterized. C-terminally to that region the protein is relatively enriched in cysteine residues, which apparently contribute to its binding of several zinc ions (Nestler et al., 2006). The amino-acid sequence in this region, however, does not strictly conform to any of the known zinc-binding motifs. A central short α-helical region in the protein binds the anti-apoptotic protein BCL-XL (Xue et al., 2002), but the function served by the cysteine-rich region (CRR) is unknown.
SIVA it is known to exist as two alternative splice isoforms or splice variants, SIVA1 and SIVA2. SIVA1 is longer and contains a death domain homology region (DDHR) with a putative amphipathical helix in its central part. SIVA2 is shorter and lacks the DDHR. Both isoforms contain a B-box-like ring finger and a Zinc finger like domain in their C-termini. Enforced expression of both SIVA1 and SIVA2 has been shown to induce apoptosis (Prasad et al., 1997, Yoon et al., 1998, Spinicelli et al., 2003, (Py et al., 2004). SIVA1 induced apoptosis is suggested to be effected by its binding to and inhibition of the anti apoptotic Bcl-2 family members through its amphipathic helical region (Chu et al., 2005; Chu et al., 2004; Xue et al., 2002). Consistent with its pro-apoptotic role, SIVA is a direct transcriptional target for tumor suppressors p53 and E2F1 (Fortin et al., 2004). Various point of evidence indicate that SIVA is a stress-induced protein and is up-regulated in acute ischemic injury (Padanilam et al., 1998), coxavirus infection (Henke et al., 2000), and also by cisplatin treatment (Qin et al., 2002), as well as TIP30 expression which induces apoptosis (Xiao et al., 2000). Recently, the common N- and C-termini of SIVA1 and SIVA2, yet not the death domain, have been shown to be sufficient and capable to mediate apoptosis in lymphoid cells through activation of a caspase dependent mitochondrial pathway (Py et al., 2004).
Recently, it was found that SIVA binds to NF-κB-inducing kinase (NIK) and controls its function (Ramakrishnan et al., 2004), has ubiquitination-related activity, is capable of directly inducing self-ubiquitination, ubiquitination of TRAF2 (a TNF-receptor associated adaptor protein 2), and that SIVA2 is an E3 ligase (WO2007080593).
Ubiquitylation, also termed ubiquitination, refers to the process particular to eukaryotes whereby a protein is post-translationally modified by covalent attachment of a small protein named ubiquitin [originally ubiquitous immunopoeitic polypeptide (UBIP)]. Ubiquitin ligase is a protein which covalently attaches ubiquitin to a lysine residue on a target protein. The ubiquitin ligase is typically involved in polyubiquitylation: a second ubiquitin is attached to the first, a third is attached to the second, and so forth. The ubiquitin ligase is referred to as an “E3” and operates in conjunction with an ubiquitin-activating enzyme (referred herein as “E1”) and an ubiquitin-conjugating enzyme (referred herein as “E2”). There is one major E1 enzyme, shared by all ubiquitin ligases, which uses ATP to activate ubiquitin for conjugation and transfers it to an E2 enzyme. The E2 enzyme interacts with a specific E3 partner and transfers the ubiquitin to the target protein. The E3, which may be a multi-protein complex, is generally responsible for targeting ubiquitination to specific substrate proteins. In some cases it receives the ubiquitin from the E2 enzyme and transfers it to the target protein or substrate protein; in other cases it acts by interacting with both the E2 enzyme and the substrate.
NIK, (MAP3K14) was discovered (Malinin et al., 1997) in a screening for proteins that bind to TRAF2. The marked activation of NF-κB upon overexpression of NIK, and effective inhibition of NF-κB activation in response to a variety of inducing agents, upon expression of catalytically inactive NIK mutants suggested that NIK participates in signaling for NF-κB activation (Malinin et al., 1997).
Assessment of the pattern of the NF-κB species in lymphoid organs indicated that, apart from its role in the regulation of NF-κB complex(s) comprised of Rel proteins and IκB, NIK also participates in controlling the expression/activation of other NF-78 B species. Indeed, NIK has been shown to participate in site-specific phosphorylation of p100, which serves as a molecular trigger for ubiquitination and active processing of p100 to form p52. This p100 processing activity was found to be ablated by the aly mutation of NIK (Xiao et al., 2001b).
NIK in thymic stroma is important for the normal production of Treg cells, which are essential for maintaining immunological tolerance. NIK mutation resulted in disorganized thymic structure and impaired production of Treg cells in aly mice (Kajiura et al., 2004). Consistently, studies of NIK-deficient mice also suggested a role for NIK in controlling the development and expansion of Treg cells (Lu et al., 2005). These findings suggest an essential role of NIK in establishing self-tolerance in a stromal dependent manner. NIK also partakes in NF-κB activation as a consequence of viral infection. Respiratory syncytial virus infection results in increased kinase activity of NIK and the formation of a complex comprised of activated NIK, IKK1, p100 and the processed p52 in alveolus like a549 cells. In this case NIK itself gets translocated into the nucleus bound to p52 and surprisingly, these events precede the activation of canonical NF-κB pathway activation (Choudhary et al., 2005). These findings indicate that NIK indeed serves as a mediator of NF-κB activation, but may also serve other functions, and that it exerts these functions in a cell- and receptor-specific manner.
NIK can be activated as a consequence of phosphorylation of the ‘activation loop’ within the NIK molecule. Indeed, mutation of a phosphorylation-site within this loop (Thr-559) prevents activation of NF-κB upon NIK overexpression (Lin et al., 1999). In addition, the activity of NIK seems to be regulated through the ability of the regions upstream and downstream of its kinase motif to bind to each other. The C terminal region of NIK downstream of its kinase moiety has been shown to be capable of binding directly to IKK1 (Regnier et al., 1997) as well as to p100 (Xiao et al., 2001b) and these interactions are apparently required for NIK function in NF-κB signaling. The N terminal region of NIK contains a negative-regulatory domain (NRD), which is composed of a basic motif (BR) and a proline-rich repeat motif (PRR) (Xiao and Sun, 2000). The N-terminal NRD interacts with the C-terminal region of NIK in cis, thereby inhibiting the binding of NIK to its substrate (IKK1 and p100). Ectopically expressed NIK spontaneously forms oligomers in which these bindings of the N-terminal to the C terminal regions in each NIK molecule are apparently disrupted, and display a high level of constitutive activity (Lin et al., 1999). The binding of the NIK C-terminal region to TRAF2 (as well as to other TRAF's) most likely participates in the activation process. However, its exact mode of participation is unknown.
Recently, a novel mechanism of NIK regulation has gained much attention. This concerns the dynamic interaction of NIK and TRAF3 leading to proteasome mediated degradation of NIK. Interestingly, inducers of the alternative pathway of NF-κB like CD40 and BLyS have been shown to induce TRAF3 degradation and concomitant enhancement of NIK expression (Liao et al., 2004).
There is rather limited information yet of the downstream mechanisms in NIK action. Evidence has been presented that NIK, through the binding of its C-terminal region to IKK1 can activate the IKB kinase (IKK) complex. It has indeed been shown to be capable of phosphorylating serine-176 in the activation loop of IKK1 and thereby its activation (Ling et al., 1998).
It was suggested that NIK does not participate at all in the canonical NF-κB pathway, but rather serves exclusively to activate the alternative one (see (Pomerantz and Baltimore, 2002, for review). However, it was lately shown that although the induction of IkappaB degradation in lymphocytes by TNF is independent of NIK, its induction by CD70, CD40 ligand, and BLyS/BAFF, which all also induce NF-kappaB2/p 100 processing, does depend on NIK function (Ramakrishnan et al. 2004). Both CD70 and TNF induce recruitment of the IKK kinase complex to their receptors. In the case of CD70, but not TNF, this process is associated with NIK recruitment and is followed by prolonged receptor association of just IKK1 and NIK. Recruitment of the IKK complex to CD27, but not that of NIK, depends on NIK kinase function. These findings indicate that NIK participates in a unique set of proximal signaling events initiated by specific inducers, which activate both canonical and noncanonical NF-kappaB dimers.
Yamamoto and Gaynor reviewed the role of NF-κB in pathogenesis of human disease (Yamamoto and Gaynor 2001). Activation of the NF-κB pathway is involved in the pathogenesis of chronic inflammatory disease, such as asthma, rheumatoid arthritis (see Tak and Firestein, this Perspective series, ref. Karin et al. 2000), and inflammatory bowel disease. In addition, altered NF-κB regulation may be involved in other diseases such as atherosclerosis (see Collins and Cybulsky, this series, ref. Leonard et al. 1995) and Alzheimer's disease (see Mattson and Camandola, this series, ref. Lin et al. 1999), in which the inflammatory response is at least partially involved. Also, abnormalities in the NF-κB pathway are also frequently seen in a variety of human cancers.
Several lines of evidence suggest that NF-κB activation of cytokine genes is an important contributor to the pathogenesis of asthma, which is characterized by the infiltration of inflammatory cells and the deregulation of many cytokines and chemokines in the lung (Ling et al. 1998). Likewise, activation of the NF-κB pathway also likely plays a role in the pathogenesis of rheumatoid arthritis. Cytokines, such as TNF-, that activate NF-κB are elevated in the synovial fluid of patients with rheumatoid arthritis and contribute to the chronic inflammatory changes and synovial hyperplasia seen in the joints of these patients (Malinin et al. 1997). The administration of antibodies directed against TNF- or a truncated TNF-receptor that binds to TNF- can markedly improve the symptoms of patients with rheumatoid arthritis.
Increases in the production of proinflammatory cytokines by both lymphocytes and macrophages have also been implicated in the pathogenesis of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis (Matsumoto et al. 1999). NF-κB activation is seen in mucosal biopsy specimens from patients with active Crohn's disease and ulcerative colitis. Treatment of patients with inflammatory bowel diseases with steroids decreases NF-κB activity in biopsy specimens and reduces clinical symptoms. These results suggest that stimulation of the NF-κB pathway may be involved in the enhanced inflammatory response associated with these diseases.
Atherosclerosis is triggered by numerous insults to the endothelium and smooth muscle of the damaged vessel wall (Matsushima et al. 2001). A large number of growth factors, cytokines, and chemokines released from endothelial cells, smooth muscle, macrophages, and lymphocytes are involved in this chronic inflammatory and fibroproliferative process (Matsushima et al. 2001). NF-□B regulation of genes involved in the inflammatory response and in the control of cellular proliferation likely plays an important role in the initiation and progression of atherosclerosis.
Also, abnormalities in the regulation of the NF-κB pathway may be involved in the pathogenesis of Alzheimer's disease. For example, NF-κB immunoreactivity is found predominantly in and around early neuritic plaque types in Alzheimer's disease, whereas mature plaque types show vastly reduced NF-□B activity (Mercurio et al. 1999). Thus, NF-κB activation may be involved in the initiation of neuritic plaques and neuronal apoptosis during the early phases of Alzheimer's disease. These data suggest that activation of the NF-κB pathway may play a role in a number of diseases that have an inflammatory component involved in their pathogenesis.
In addition to a role in the pathogenesis of diseases characterized by increases in the host immune and inflammatory response, constitutive activation of the NF-κB pathway has also been implicated in the pathogenesis of some human cancers. Abnormalities in the regulation of the NF-κB pathway are frequently seen in a variety of human malignancies including leukemias, lymphomas, and solid tumors (Miyawaki et al. 1994). These abnormalities result in constitutively high levels of NF-κB in the nucleus of a variety of tumors including breast, ovarian, prostate, and colon cancers. The majority of these changes are likely due to alterations in regulatory proteins that activate signaling pathways that lead to activation of the NF-κB pathway. However, mutations that inactivate the I B proteins in addition to amplification and rearrangements of genes encoding NF-κB family members can result in the enhanced nuclear levels of NF-κB seen in some tumors.
Apart from the contribution to the regulation of the development and function of the immune system, NIK seems also to be involved in the regulation of various non-immune functions such as mammary gland development (Miyawaki et al., 1994). NIK has a role in lymphoid organ development (Shinkura et al., 1999). In vitro studies implicated NIK in signaling that leads to skeletal muscle cell differentiation (Canicio et al., 2001), and in the survival and differentiation of neurons (Foehr et al., 2000).
A need of a satisfactory treatment exists for numerous lethal and/or highly debilitating diseases associated with disregulated activity of NIK and/or NF-.κ.B molecules, including malignant diseases and diseases associated with pathological immune responses, such as autoimmune, allergic, inflammatory, and transplantation-related diseases.